Name: full-circle cauterization of limbal vascular plexus for surgically induced glaucoma in rodents
Uploaded: 2022-02-15T14:00:00
Description: Watch this Scientific Journal Video about Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents at JoVE.com

We acknowledge our laboratory technicians Jos&#233;; Nilson dos Santos, Daianne Mandarino Torres, Jos&#233; Francisco Tib&#250;rcio, Gildo Brito de Souza, and Luciano Cavalcante Ferreira. This research was funded by FAPERJ, CNPq, and CAPES.

All procedures were performed in compliance with the Statement for the Use of Animals in Ophthalmic and Visual Research from the Association for Research in Vision and Ophthalmology (ARVO) and approved by the Ethics Committee on the Use of Animals in Scientific Experimentation from the Health Sciences Center, Federal University of Rio de Janeiro (protocol 083/17). In the present work, Lister Hooded rats of both genders were used, aged 2-3 months and weighing 180-320 g. However, the procedure can be adapted in different r...

<ol>
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Limbal plexus cauterization (LPC) is a novel post-trabecular model with the advantage that it targets easily accessible vascular structures not requiring conjunctival or tenon dissection17,28. Differently from the vortex veins cauterization model, a renowned OHT model based on the surgical impairment to choroid venous drainage, venous congestion is not expected to influence IOP rise in the LPC model, as limbal veins are situated upstream in aqueous humor outflow....

Medical literature understands glaucoma as a heterogeneous group of optic neuropathies characterized by progressive degeneration of retinal ganglion cells (RGCs), dendrites, soma, and axons, resulting in structural cupping (excavation) of the optic disc and functional deterioration of the optic nerve, leading to amaurosis in uncontrolled cases by interrupting the transmission of visual information from the eye to the brain1. Glaucoma is currently the most common cause of irreversible blindness worldwide, predicted to reach approximately 111.8 million people in 20402, thus deeply affecting patients&#39; quality of life (QoL) and leading to significant socioeconomic concerns3.
Elevated intraocular pressure (IOP) is one of the most important and the only modifiable risk factor for the development and progression of glaucoma. Among the multiple types of glaucoma, all, except for normal tension glaucoma (NTG), are associated with elevated IOP at some time in the clinical history of the disease. Despite remarkable clinical and surgical advances to target IOP and slow down or stop disease progression, patients still lose sight due to glaucoma4,5. Therefore, a thorough understanding of the complex and multifactorial pathophysiology of this disease is imperative for the development of more effective treatments, especially to provide neuroprotection to RGCs.
Among a variety of experimental approaches for the understanding of disease mechanisms, animal models based on ocular hypertension (OHT) most closely resemble human glaucoma. Rodent models are particularly useful as they are&#160;low-cost, are easy to handle,&#160;can be genetically manipulated, have a short lifespan, and present ocular anatomical and physiological features comparable to humans, such as aqueous humor production and drainage6,7,8,9,10,11,12,13. Currently used models include sclerosis of the trabecular meshwork following injection of hypertonic saline into episcleral veins14, intracameral injection of microbeads15&#160;or viscoelastic substances16, cauterization of vortex veins17, photocoagulation of the trabecular meshwork with argon laser18, circumlimbal suture19, and use of a transgenic model of age-related OHT (DBA/2J mice)8. However, invasiveness, post-operative opacification of the cornea, anterior segment disruption, extensive learning curves, expensive equipment, and highly variable postoperative IOPs, are among few of the reported pitfalls associated with the current models, making the development of an alternative model of OHT a demand to overcome these problems20,21,22.
The present protocol formalizes a novel surgical procedure to induce OHT as a proxy to glaucoma, based on limbal plexus cauterization (LPC) in rodents23. This is an easy, reproducible, accessible, and non-invasive model that provides high efficiency and low variability of IOP elevation, associated with a uniquely high rate of full clinical recovery, therefore providing in vivo functional evaluation in a reduced number of animals used in each experiment. The surgery technique induces subacute OHT with a gradual return to baseline levels in a few days, which models the hypertensive attack seen in acute angle-closure glaucoma. Moreover, the IOP recovery in the model is followed by continuous glaucomatous neurodegeneration, which is useful for future mechanistic studies of the secondary degeneration of RGCs, which occurs in several cases of human glaucoma despite adequate control of IOP.

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			<td>Acetone</td>
			<td>Isofar</td>
			<td>201</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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		<tr>
			<td>Active electrode for electroretinography</td>
			<td>Hansol Medical Co</td>
			<td>-</td>
			<td>Stainless steel needle 0.25 mm &#215; 15 mm</td>
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			<td>Anestalcon</td>
			<td>Novartis Bioci&#234;ncias S/A</td>
			<td>MS-1.0068.1087</td>
			<td>Proxymetacaine hydrochloride 0.5%</td>
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			<td>Calcium chloride</td>
			<td>Vetec</td>
			<td>560</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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			<td>Cautery Low Temp Fine Tip 10/bx</td>
			<td>Bovie Medical Corporation</td>
			<td>AA00</td>
			<td>Low-temperature ophthalmic cautery</td>
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			<td>Syntec do Brasil Ltda</td>
			<td>000200-3-000003</td>
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			<td>DAKO</td>
			<td>Dako North America</td>
			<td>S3023</td>
			<td>Antifade mounting medium</td>
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			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>28718-90-3</td>
			<td>diamidino-2-phenylindole; blue fluorescent nuclear counterstain; emission at 452&#177;3 nm</td>
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			<td>Ecofilm</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0298.0487</td>
			<td>Carmellose sodium 0.5%</td>
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			<td>EPON Resin</td>
			<td>Polysciences, Inc.</td>
			<td>-</td>
			<td>Epoxy resin used for electron microscopy, composed of a mixture of four reagents: Poly/Bed 812 Resin (CAT#08791); DDSA - Dodecenylsuccinic Anhydride (CAT#00563); NMA - Nadic Methyl Anhydride (CAT#00886); DMP-30 - 2,4,6-tris(dimethylaminomethyl)phenol (CAT#00553)</td>
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			<td>Glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16110</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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			<td>Hyabak</td>
			<td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td>
			<td>MS-8042140002</td>
			<td>Sodium hyaluronate 0.15%</td>
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			<td>Icare Tonolab</td>
			<td>Icare Finland Oy</td>
			<td>TV02 (model number)</td>
			<td>Rebound handheld tonometer</td>
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			<td>IgG donkey anti-mouse antibody + Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific</td>
			<td>A31570</td>
			<td>Secondary antibody solution</td>
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			<td>LCD monitor 23 inches</td>
			<td>Samsung Electronics Co. Ltd.</td>
			<td>S23B550</td>
			<td>Model LS23B550, for electroretinogram recording</td>
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			<td>LSM 510 Meta</td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>Confocal epifluorescence microscope</td>
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			<td>Maxiflox</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0298.0489</td>
			<td>Ciprofloxacin 3.5 mg/g</td>
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			<td>MEB-9400K</td>
			<td>Nihon Kohden Corporation</td>
			<td>-</td>
			<td>System for electroretinogram recording</td>
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			<td>monoclonal IgG1 mouse anti-Brn3a</td>
			<td>MilliporeSigma</td>
			<td>MAB-1585</td>
			<td>Brn3a primary antibody solution</td>
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			<td>Neuropack Manager v08.33</td>
			<td>Nihon Kohden Corporation</td>
			<td>-</td>
			<td>Software for electroretinogram signal processing</td>
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			<td>Optomotry</td>
			<td>CerebralMechanics</td>
			<td>-</td>
			<td>System for optomotor response analysis</td>
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			<td>Osmium tetroxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>19100</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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			<td>Potassium ferrocyanide</td>
			<td>Electron Microscopy Sciences</td>
			<td>20150</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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			<td>Reference and ground electrodes for electroretinography</td>
			<td>Chalgren Enterprises</td>
			<td>110-63</td>
			<td>Stainless steel needles 0.4 mm &#215; 37 mm</td>
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			<td>Sodium cacodylate buffer</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
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		<tr>
			<td>Ster MD</td>
			<td>Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A</td>
			<td>MS-1.0497.1287</td>
			<td>Prednisolone acetate 0.12%</td>
		</tr>
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			<td>Terolac</td>
			<td>Crist&#225;lia Produtos Qu&#237;micos Farmac&#234;uticos Ltda</td>
			<td>MS-1.0497.1286</td>
			<td>Ketorolac trometamol 0.5%</td>
		</tr>
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			<td>Terramicina</td>
			<td>Laborat&#243;rios Pfizer Ltda</td>
			<td>MS-1.0216.0024</td>
			<td>Oxytetracycline hydrochloride 30 mg/g + polymyxin B 10,000 U/g</td>
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			<td>Tono-Pen XL</td>
			<td>Reichert Technologies</td>
			<td>230635</td>
			<td>Digital applanation handheld tonometer</td>
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			<td>TO-PRO-3</td>
			<td>Thermo Fisher Scientific</td>
			<td>T3605</td>
			<td>Far red-fluorescent nuclear counterstain; emission at 661 nm</td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9036-19-5</td>
			<td>Non-ionic surfactant</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>Used for electron microscopy tissue preparation (step 5)</td>
		</tr>
		<tr>
			<td>Xilazin</td>
			<td>Syntec do Brasil Ltda</td>
			<td>7899</td>
			<td>Xylazine hydrochloride 2%</td>
		</tr>
		<tr>
			<td></td>
			<td>Carl Zeiss</td>
			<td>-</td>
			<td>Stereo microscope for surgery and retinal dissection</td>
		</tr>
	</tbody>
</table>

full-circle cauterization of limbal vascular plexus for surgically induced glaucoma in rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The quantitative variables are expressed as mean &#177; standard error of the mean (SEM). Except for the comparison of IOP dynamics between OHT and control groups (Figure 1F), statistical analysis was performed using two-way ANOVA followed by Sidak&#39;s multiple comparisons test. A p-value &#60; 0.05 was considered statistically significant.
Figure 1 illustrates surgical steps of the full-circle limbal plexus cauterization (LPC) mode...

Watch this Scientific Journal Video about Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents at JoVE.com

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

We developed a simple method to induce robust and progressive glaucoma degeneration based on temporary intraocular pressure elevation. This represents a unique opportunity to study key points of glaucoma pathophysiology. This is an easy-learning, non-invasive, low-cost, and highly-reproducible model that enables in vivo function analysis and short-term experimental designs using a reduced number of animals for each experiment.The retina and optic nerve are assessable parts of the central nervous system, so modeling the progressive impairment of these structures can provide valuable insights into other neurodegenerative disorders. To measure baseline intraocular pressure, or IOP, of both experimental and contralateral control eyes, place the anesthetized rat in a ventral decubitus position on the bench top, such that the corneal surface is easily accessible to the tip of the tonometer. Position the tonometer tip such that it slightly touches the central corneal zone perpendicularly.Then, acquire the measurement by pressing the measurement button. Next, place the rat in a slight lateral decubitus position under a stereo microscope and inspect the experimental eye at 40 times magnification. During the procedure, keep the contralateral control eye lubricated by instilling a drop of carmellose sodium or sodium hyaluronate.Next, using curved forceps, gently push the experimental eyeball forward to expose the vasculature surrounding 360 degrees of the limbus. Then, with the other hand, using a low-temperature ophthalmic cautery, gently cauterize the vessels all around the cornea. Be careful not to cauterize the corneal periphery.Confirm the success of the surgical procedure by observing the emergence of small circular marks of cauterization on the scleral limbus, the obliteration of limbal vasculature, and pupil dilation in the operated eye. After the surgery, check the immediate postoperative IOP in both eyes as demonstrated earlier. Then, apply a drop of ophthalmic prednisolone acetate to the anterior surface of the experimental eye.After 40 seconds, replace it with an ophthalmic antibiotic ointment. Apply a daily clinical follow-up of the experimental eye with topical medications composed of a non-steroidal anti-inflammatory drug and antibiotic ointment. For optomotor response analysis, place the rat for habituation on a platform built with four computer monitors in a quadrangle.Maintain the red cross-hair cursor on the video frame between the eyes of the freely moving rat, as it indicates the center of the virtual cylinder. Observe the optomotor response consisting of a reflexive tracking of the rat head and neck elicited by the rotating gradings. Initially, introduce a stimulus with low spatial frequency, then increase the frequency progressively until tracking movement is not noticed.To record the pattern electroretinogram, after anesthetizing the rat, carefully insert the active electrode at the temporal periphery of the cornea. Additionally, insert the reference electrode into the subcutaneous tissue of the ipsilateral temporal canthus and the ground electrode into one of the hind limbs. Then position the rat 20 centimeters from the stimulus screen, monitor the signal baseline, and start the acquisition by pressing the Analysis button on the acquisition system.After isolating the retinas, transfer them into a 24-well culture plate containing one-milliliter PBS, keeping the inner retina facing up. Permeabilize the tissue by washing with a 0.5%non-ionic surfactant diluted in PBS. Then, incubate the tissue and blocking solution for one hour at room temperature with gentle shaking.During the incubation, prepare Brn-3a primary antibody solution and store at four degree Celsius. After tissue blocking, incubate the retinas in 200 microliters of primary antibody solution at four degrees Celsius for 72 hours with gentle shaking. Next, wash the tissue with PBS.Incubate the tissue in 200 microliters of the secondary antibody solution for two hours at room temperature and then with nuclear counter stain solution for 10 minutes for fluorescent nuclei staining. After three PBS washes, transfer the retina onto a glass microscope slide using two small brushes maintaining the vitreous side up. Position the dorsal retinal quadrant up on the microscope slide.Finally, apply 200 microliters of antifade mounting medium on a glass cover slip and place it onto the flat mounted retina for microscopic analysis. Under a confocal epifluorescence microscope, using a 40 times magnifying objective, examine the flat mounts to estimate retinal ganglion cells. After eyeball enucleation, remove the proximal segment of the intraorbital portion of the optic nerve, including part of the intraocular portion.Then, immediately place the samples into vials or tubes containing 200 to 300 microliters of cold fixative. After two hours, wash the tissue with cold 100 micromolar sodium cacodylate buffer. Then, post-fixate the tissue for one hour in 1%osmium tetroxide and five nanomolar calcium chloride at four degrees Celsius under gentle shaking.After three washes with cold sodium cacodylate buffer, wash the optic nerve fragments with cold distilled water before incubating overnight and 1%urinal acetate at four degrees Celsius. The following day, after three washes with water, progressively dehydrate the tissue in increasing acetone concentrations. Then, perform three rounds of embedding and epoxy resin acetone solutions before embedding the tissue in pure epoxy resin for 24 hours.The next day, remove the samples from the specimen carrier and transfer them to embedding molds for 48 hours at 60 degrees Celsius. After polymerization, use an ultra microtome to cut transversal semi-thin sections of the optic nerve fragments. Then, collect and transfer the sections onto a microscope glass slide.Stain the sections with toluidine blue and image them using an optic microscope at 100 times magnification. For ultra structural analysis, perform ultra thin cross-sections and collect them on a copper grid. Then, stain the sections with urinal acetate and lead citrate for transmission electron microscopic examination.Full-circle cauterization induced IOP elevation immediately after surgery compared to control eyes. The peak for postoperative IOP was observed on day one and decreased gradually to the baseline levels on day six. Further, retinal function was evaluated both behaviorally and electrophysiologically using optomotor reflex and pattern electroretinogram, respectively.After the surgery, both parameters showed two distinct phases of impairment:ocular hypertension acute phase on day three and a secondary degeneration phase on day 30. Compared to control optic nerves, axonal counts and semi-thin transversal optic nerve sections decreased progressively after surgery. Transmission electron micrographs of optic nerves from the control eye demonstrated densely-packed myelinated fibers separated by thin glial cell processes and evident axonal microtubules and neurofilaments.In contrast, after three days of ocular hypertension, a few degenerated fibers, cytoplasmic vacuolation in glial cell processes, and condensed chromatin in glial cell nuclei were observed. After seven days, there was an increase in degenerated axonal fibers and hypertonic glial cell processes. And after 14 days, more disarrangement of the optic nerve fibers associated with the invasion of glial cell processes among the axons were observed.Furthermore, the density of retinal ganglion cells decreased with time mainly in the dorsal and temporal retinal quadrants. While performing this procedure, try to gently touch limbal vessels with a cautery tip, sparing the cord periphery and ensuring continuous cauterization marks are observed all around the scleral limbus. Following this procedure, a variety of methods can be applied, such as functional biochemical and immunohistochemical evaluation of eye structures and other methods that may acknowledge on glaucoma physiopathology.

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